Novel method for generation of rna virus

ABSTRACT

The present invention provides a method for generating negative-stranded segmented RNA viruses using linear expression constructs in the presence of helper virus.

The present invention provides a method for generating negative-strandedsegmented RNA viruses using linear expression constructs in the presenceof helper virus.

BACKGROUND OF THE INVENTION

Negative-strand RNA viruses are a group of animal viruses that compriseseveral important human pathogens, including influenza, measles, mumps,rabies, respiratory syncytial, Ebola and hantaviruses.

The genomes of these RNA viruses can be unimolecular or segmented, andare single stranded of (−) polarity. Two essential requirements areshared between these viruses: their genomic RNAs must be efficientlycopied into viral RNA, a form which can be used for incorporation intoprogeny virus particles and transcribed into mRNA which is translatedinto viral proteins. Eukaryotic host cells typically do not contain themachinery for replicating RNA templates or for translating polypeptidesfrom a negative-stranded RNA template. Therefore negative-strand RNAviruses encode and carry an RNA-dependent RNA polymerase to catalyzesynthesis of new genomic RNA for assembly into progeny viruses and mRNAsfor translation into viral proteins.

Genomic viral RNA must be packaged into viral particles in order for thevirus to be transmitted. The processes by which progeny viral particlesare assembled and the protein/protein interactions that occur duringassembly are similar within the RNA viruses. The formation of virusparticles ensures the efficient transmission of the RNA genome from onehost cell to another within a single host or among different hostorganisms.

Virus families containing enveloped, single-stranded RNA with anegative-sense genome are classified into groups having non-segmentedgenomes (Paramyxoviridae, Rhabdoviridae, Filoviridae and Borna DiseaseVirus, Togaviridae) and those having segmented genomes(Orthomyxoviridae, Bunyaviridae and Arenaviridae). The Orthomyxoviridaefamily includes the viruses of influenza, types A, B and C viruses, aswell as Thogoto and Dhori viruses and infectious salmon anemia virus.

Influenza virions consist of an internal ribonucleoprotein core (ahelical nucleocapsid) containing the single-stranded RNA genome, and anouter lipoprotein envelope lined inside by a matrix protein (M1). Thesegmented genome of influenza A virus consists of eight molecules oflinear, negative polarity, single-stranded RNAs which encode elevenpolypeptides (ten in some influenza A strains), including: theRNA-dependent RNA polymerase proteins (PB2, PB1 and PA) andnucleoprotein (NP) which form the nucleocapsid; the matrix membraneproteins (M1, M2); two surface glycoproteins which project from thelipid-containing envelope: hemagglutinin (HA) and neuraminidase (NA);the nonstructural protein (NS1) and nuclear export protein (NEP). Mostinfluenza A strains also encode an eleventh protein (PB1-F2) believed tohave proapoptotic properties.

Transcription and replication of the viral genome takes place in thenucleus and assembly occurs via budding on the plasma membrane. Theviruses can reassort genes during mixed infections. Influenza virusadsorbs via HA to sialyloligo-saccharides in cell membrane glycoproteinsand glycolipids. Following endocytosis of the virion, a conformationalchange in the HA molecule occurs within the cellular endosome whichfacilitates membrane fusion, thus triggering uncoating. Thenucleo-capsid migrates to the nucleus where viral mRNA is transcribed.Viral mRNA is transcribed by a unique mechanism in which viralendonuclease cleaves the capped 5′-terminus from cellular heterologousmRNAs which then serve as primers for transcription of viral RNAtemplates by the viral transcriptase. Transcripts terminate at sites 15to 22 bases from the ends of their templates, where oligo(U) sequencesact as signals for the addition of poly(A) tracts. Of the eight viralRNA molecules so produced, six are monocistronic messages that aretranslated directly into the proteins representing HA, NA, NP and theviral polymerase proteins, PB2, PB1 and PA. The other two transcriptsundergo splicing, each yielding two mRNAs which are translated indifferent reading frames to produce M1, M2, NS1 and NEP. In other words,the eight viral RNA segments code for eleven proteins: nine structuraland 2 non-structural (NS1 and the recently identified PB1-F2) proteins.

The generation of modern vaccines for influenza viruses, especially forhighly pathogenic avian influenza viruses, relies on the use of reversegenetics, which allows the production of influenza viruses from DNA. Thefirst reverse genetic systems for construction of negative-strand RNAinfluenza viruses involved the transfection of a single viral gene mixedwith in-vitro reconstituted ribonucleoprotein (RNP) complexes andsubsequent infection with an influenza helper virus. RNP complexes weremade by incubating synthetic RNA transcripts with purified NP andpolymerase proteins (PB1, PB2 and PA) from influenza viruses, and ahelper virus was used as an intracellular source of viral proteins andof the other vRNAs (Luytjes et al., 1989, Cell, 59, 1107-1113).

Neumann et al. (1994, Virology, 202, 477-479) achieved RNP formation ofviral model RNAs in influenza-infected cells after expression of RNAfrom a murine RNA polymerase I promoter-responsive plasmid. Pleschka etal. (1996, J. Virol., 4188-4192) described a method wherein RNPcomplexes were reconstituted from plasmid-based expression vectors.Expression of a viral RNA-like transcript was achieved from a plasmidcontaining a truncated human polymerase I (polI) promoter and a ribozymesequence that generated a 3″end by autocatalytic cleavage. Thepoll-driven plasmid was cotransfected into human 293 cells withpolII-responsive plasmids that expressed the viral PB1, PB2, PA and NPproteins. Transfection efficiency was very low, however, withapproximately 10 transfectant virus particles per transfection.Additionally, this plasmid-based strategy was dependent on the aid of ahelper virus.

In WO 01/04333, segmented negative-strand RNA viruses were constructedusing a set of 12 expression plasmids for expressing genomic vRNAsegments and RNP proteins. The vectors described in WO 01/04333 werebased on well known pUC19 or pUC18 plasmids. According to thedescription, this system requires a set of 8 plasmids expressing all 8segments of influenza virus together with an additional set of 4plasmids expressing nucleoprotein and subunits of RNA-dependent RNApolymerase (PB1, PB2, PA and NP).

WO 00/60050 covers a set of at least two vectors comprising a promoteroperably linked to an influenza virus segment cDNA (PA, PB1, PB2, HA,NP, NA, M) and linked to a transcription termination sequence, and atleast two vectors comprising a promoter operably linked to an influenzavirus segment DNA (PA, PB1, PB2, NP). This system attempted to overcomethe difficulties in using of a large number of different vectors byusing plasmids with eight RNA polymerase I transcription cassettes forviral RNA synthesis combined on one plasmid.

WO 01/83794 discloses circular expression plasmids comprising an RNApolymerase I (polI) promoter and a polI termination signal, insertedbetween a RNA polymerase II (polII) promoter and a polyadenylationsignal. The term vector according to this application is described as aplasmid which generally is a self-contained molecule of double-strandedDNA that can accept additional foreign DNA and which can be readilyintroduced into a suitable host cell.

WO 2009/00891 describes a linear expression construct and its use forexpression of influenza virus gene segments.

Ozawa M. et al (J. Virol, 2007, vol. 81, pp. 9556-9559) describes areverse genetics system for the generation of influenza A virus usingadenovirus vectors. Hoffmann E. et al (Virology, 2000, 267, pp. 310-317)disclose a system for creating influenza virus by generating viral RNAand mRNA from one template using a bidirectional transcriptionconstruct. The rescue of influenza B virus from eight plasmids was alsodisclosed in Hoffmann et al. (Proc. Natl. Acad. Sci., 2002, 99, pp.11411-11416).

Epidemics and pandemics caused by viral diseases are still claiminghuman lives and are impacting the global economy. Influenza isresponsible for millions of lost work days and visits to the doctor,hundreds of thousands of hospitalizations worldwide (Couch 1993, Ann.NY. Acad. Sci 685; 803), tens of thousands of excess deaths (Collins &Lehmann 1953 Public Health Monographs 213:1; Glezen 1982 Am. J. PublicHealth 77:712) and billions of Euros in terms of health-care costs(Williams et al. 1988, Ann. Intern. Med. 108:616). When healthy adultsget immunized, currently available vaccines prevent clinical disease in70-90% of cases. This level is reduced to 30-70% in those over the ageof 65 and drops still further in those over 65 living in nursing homes(Strategic Perspective 2001: The Antiviral Market. Datamonitor. p. 59).The virus's frequent antigenic changes further contribute to a largedeath toll because not even annual vaccination can guarantee protection.Hence, the U.S. death toll rose from 16,363 people in 1976/77 to fourtimes as many deaths in 1998/99 (Wall Street Journal, Flu-related deathsin US despite vaccine researches. Jan. 7, 2003).

Especially in case of the outbreak of pandemic viral diseases, it can beof utmost importance to provide vaccinations or treatments immediatelyafter outbreak of the disease. In view of the urgent need for providingefficient protection and treatment of viral diseases there is a stillhigh demand for the development of economic, fast and efficientexpression systems for virus production which can overcome thedisadvantages and difficulties of the present expression technologiesand provide an alternative method for virus expression. The object isachieved by the provision of the embodiments of the present application.

BRIEF SUMMARY OF THE INVENTION

The present invention provides an alternative technology wherein linearexpression constructs are used for expression of RNA viruses in thepresence of a helper virus.

It has been surprisingly found that the use of at least one linearexpression construct free of any amplification and/or selectionsequences comprising an RNA polymerase I (polI) promoter and a polItermination signal, inserted between an RNA polymerase II (polII)promoter, and a polyadenylation signal comprising a HA or a NA genesegment which is inserted between the polI promoter and the polItermination signal, in the presence of a helper virus provides anefficient tool for fast rescue of viral particles. In contrast to themethods used by known technologies, no cloning steps in bacterial cellsare needed and host cells need not be transfected with all segments ofthe viral genome. Specifically, transfection with only one or twosegments, i.e. genes coding for the HA and/or NA protein, can besufficient for expression of whole virus. Therefore, the time needed fortransfection and expression of sufficient amounts of viral particles canbe highly reduced.

For example, a linear expression construct as described inPCT/EP2008/058182, which is incorporated herein by reference, can beused for developing vaccines comprising RNA viruses, specificallyinfluenza viruses either of wild type, mutant or reassortant strains, inthe presence of helper virus. This provides a tool for fast generationof any virus vaccine needed in case of the occurrence of influenzaepidemics or pandemics.

Further, the present invention provides an improved method for removalof helper virus and provides HA segments with modified cleavage sitesfor improved selection and purification purposes.

FIGURES

FIGS. 1 a and 1 b is a schematic diagram illustrating the generation oflinear bidirectional expression constructs. FIG. 1 a shows fragments F1,F2 and F3 being generated separately by PCR amplification. FIG. 1 bshows fragment F4 being generated by overlapping PCR using theoligonucleotides P4 and P6.

DETAILED DESCRIPTION OF THE INVENTION

The present invention covers a method for production of negativestranded segmented RNA viruses comprising the steps of

a) providing a linear expression construct free of any amplificationand/or selection sequences, which construct comprises an RNA polymeraseI (polI) promoter and a poll termination signal, both inserted betweenan RNA polymerase II (polII) promoter and a polyadenylation signal whichconstruct further comprises a HA and/or a NA gene segment insertedbetween the polI promoter and the polI termination signal,b) transfecting a host cell with said linear expression construct,c) infecting said host cells with a helper virus having helper virus HAand/or NA proteins,d) cultivating said host cell to propagate virus particles,e) selecting virus particles, which contain

-   -   (i) the HA and/or NA proteins derived from the linear expression        construct, but not    -   (ii) the helper virus HA and NA proteins, or segments thereof,

wherein said selection is based on phenotypic, genotypic or antigenicproperties of the HA and/or NA proteins, and optionally

wherein the absence of helper virus HA and NA proteins is determined byanalysis of the nucleic acid or amino acid sequence.

More specifically the method for producing a negative-stranded,segmented RNA virus particle can comprise the steps of providing alinear expression construct free of amplification sequences, selectionsequences, or both amplification sequences and selection sequences,wherein the construct comprises an RNA polymerase I (polI) promoter anda polI termination signal, the polI promoter and polI termination signalbeing inserted between an RNA polymerase II (polII) promoter and apolyadenylation signal, wherein the linear expression construct furthercomprises an HA gene segment, an NA gene segment, or both an HA genesegment and an NA gene segment inserted between the polI promoter andthe polI termination signal; transfecting a host cell with the linearexpression construct; infecting the host cell with a helper virus,wherein the helper virus comprises genomic RNA encoding HA protein, NAprotein or both HA protein and NA protein; cultivating the host cell,thereby producing progeny virus particles, wherein at least some of theprogeny virus particles comprise HA protein or NA protein derived fromthe linear expression construct; and selecting a candidate virusparticle from among the progeny virus particles, wherein the candidatevirus particle comprises:

i) HA protein derived from the linear expression construct and not HAprotein derived from the helper virus, if the linear expressionconstruct comprises an HA gene segment; andii) NA protein derived from the linear expression construct and not NAprotein derived from the helper virus, if the linear expressionconstruct comprises an NA gene segment.

According to invention the host cell is transfected with at least onelinear expression construct comprising an HA or NA gene segment.Preferably the host cell is transfected with at least two linearexpression constructs wherein one linear construct comprises the HA genesegment and the second linear construct comprises the NA gene segment.

The step of selecting the candidate virus particle can further compriseanalyzing amino acid sequences of the candidate virus particle in orderto determine that the candidate virus particle does not comprise HAamino acid sequences or NA amino acid sequences of the helper virus oranalyzing nucleic acid molecules of the candidate virus particle inorder to determine that the candidate virus particle does not compriseHA nucleotide sequences or NA nucleotide sequences of the helper virus.

The “linear expression constructs” are defined according to theinvention as being free of any amplification and/or selection sequencesand comprising an RNA polymerase I (polI) promoter and a polItermination signal, inserted between an RNA polymerase II (polII)promoter and a polyadenylation signal and comprising a gene segmentinserted between the polI promoter and the polI termination signal.

Preferably, the linear expression constructs do not contain anyselection or amplification sequences that are needed for amplificationof plasmids in bacterial cells. Neither on (origin ofreplication)-sequences nor antibiotics resistance genes or any otherselection markers need to be contained. If needed, the linear expressionconstruct can be circularized using short linker sequences.

According to a specific embodiment of the invention the linearexpression construct can comprise molecules other than DNA molecules,such as additional protection sequences at the N- and/or C-terminus ofthe construct. For example, these protection sequences can be peptidenucleic acid sequences (PNAs) as described in WO 00/56914. These PNAsare nucleic acid analogs in which the entire deoxyribose-phosphatebackbone has been exchanged with a chemically completely different, butstructurally homologous, polyamide (peptide) backbone containing2-aminoethyl glycine units. PNA “clamps” have also been shown toincrease stability, wherein two identical PNA sequences are joined by aflexible hairpin linker containing three 8-amino-3,6-dioxaoctanoic acidunits. When a PNA is mixed with a complementary homopurine orhomopyrimidine DNA target sequence, a PNA-DNA-PNA triplex hybrid canform which is extremely stable (Bentin et al., 1996, Biochemistry, 35,8863-8869, Egholm et al., 1995, Nucleic Acids Res., 23, 217-222, Nielsenet al., Science, 1991, 254, 1497-1500, Demidov et al., Proc. Natl. Acad.Sci., 1995, 92, 2637-2641). They have been shown to be resistant tonuclease and protease digestion (Demidov et al., Biochem. Pharm., 1994,48, 1010-1013). The viral gene segment can be a cDNA copy or RT-PCRamplification product of said segment.

Specifically, the present invention provides a method for expression andproduction of an a RNA virus comprising the steps of

a) transfecting host cells with a linear expression construct comprisingan HA gene segment and/or a linear expression construct comprising an NAgene segment and optionally linear expression constructs comprisingfurther gene segments or at least part thereof selected from PB1, PB2,PA, NS, M, NPb) infecting said host cells with a helper virusc) cultivating the infected host cells to propagate virusesd) selecting virus particles containing at least HA and/or NA proteinderived from said linear expression constructs.

Said selection can be performed based on genotypic, phenotypic orantigenic properties of the HA or NA proteins of non-helper virusorigin. Any selection methods can be used as known to differentiatebetween proteins comprising different sequences, different phenotypiccharacteristics or different antigenic characteristics. Specifically,selection criteria can be used as described in the present invention.The HA and NA proteins from helper virus origin and non-helper virusorigin vary in nucleotide and amino acid sequence, therefore sequencescomparison methods as well known in the art can be used for identifyingviruses comprising HA or NA sequences derived from the linear expressionconstructs. Nucleic acid molecules that are “derived from” an expressionconstruct or a virus are those that comprise a nucleotide sequence ofthe expression construct or virus or a complementary sequence, and aregenerally produced as a result of the presence of the expressionconstruct or virus in a cell culture or other medium for production ofthe molecules. Proteins that are “derived from” an expression constructor a virus are those which are translated from a nucleotide sequence ofthe expression construct or virus or a complementary sequence, and aregenerally produced as a result of the presence of the expressionconstruct or virus in a cell culture or other medium for production ofthe proteins.

Additionally to the use of at least one linear expression construct,plasmids known in the art for performing reverse genetics techniques canbe used for expression of viral proteins and/or further segments of theviral genome. These plasmids are for example described in Hoffmann etal. (Vaccine 2002, 20(25-26), 3165-3170, which is incorporated byreference). Specifically, these expression plasmids comprise thesegments coding for PB1, PB2, PA, NS, NA, HA, M or NP or part thereof.

The term “HA protein and NA protein” are defined according to thepresent invention as the complete amino acid sequence of the HA or NAprotein respectively or a part of said sequence wherein said part issufficient to induce an immune response against said HA or NA proteinsimilar or equal to the response produced by wild type HA or NA protein.Preferably, the HA or NA protein comprises at least 70% of the HA or NAamino acid sequence of the complete protein, preferably at least 90%,more preferably at least 95%,

Functional equivalent in terms of immunogenicity can be tested forexample in animal models as described in Lu et al. (J. Virol., 1999,5903-5911) or Boyd M. R. and Beeson M. F. (J. AntimicrobialChemotherapy, 1975, 43-47)

A helper virus is a virus used when producing copies of a helperdependent viral vector which does not have the ability to replicate onits own. The helper virus is used to coinfect cells alongside the viralvector and provides the necessary enzymes for replication of the genomeof the viral vector.

The term “helper virus” is defined as any virus that comprises at leastone gene segment identical to the virus to be produced and which cansupport the virus generation by providing at least one viral segmentand/or at least one viral protein needed for producing complete virusparticles.

The helper virus is generally added to the host cells in the presentmethod after transfection with linear expression constructs, yetaccording to an alternative method, the helper virus can be added to thehost cells for infection before the host cells are transfected by theexpression construct comprising HA and/or NA gene segments.

The RNA viruses that can be expressed by said method can be any RNAvirus comprising HA and/or NA gene segments or structures functionallyequivalent to these structures. The term “functionally equivalentstructures” means viral proteins that have receptor-binding and fusionactivities.

The RNA viruses can be selected from the group consisting of influenzaviruses, specifically influenza A, B or C viruses, coronavirus,Respiratory Syncytial virus, Newcastle disease virus.

The cells which can be used in the method according to the invention forcultivating the viruses can be any desired type of cells which can becultured and which can be infected by enveloped viruses, specifically byinfluenza viruses. Specifically it can be BSC-1 cells, LLC-MK cells,CV-1 cells, CHO cells, COS cells, murine cells, human cells, HeLa cells,293 cells, VERO cells, CEK (chicken embryo kidney) CEF (chicken embryofibroblasts), MDBK cells, MDCK cells, MDOK cells, CRFK cells, RAF cells,TCMK cells, LLC-PK cells, PK15 cells, WI-38 cells, MRC-5 cells, T-FLYcells, BHK cells, SP2/0 cells, NS0, PerC6 (human retina cells).

According to the inventive method the host cells can be transfected byknown methods, for example by electroporation.

The host cell culture can be cultured under standard conditions known inthe art to replicate the viruses, in particular until a maximumcytopathic effect or a maximum amount of virus antigen can be detected.The harvesting can alternatively be at any timepoint during cultivation.

The pH for cultivation of the host cells, can be for example between 6.5and 7.5. The pH for cultivation depends on the pH stability of the hostcells used for cultivation. This can be determined by testing of thehost cells' viability under different pH conditions.

It is well known in the art that the wild-type viruses used inpreparation of the vaccine strains for annual vaccination againstepidemic influenza are recommended annually by the World HealthOrganization (WHO). These strains may then used for the production ofreassortant vaccine strains which generally combine the NA and/or HAgenes of the wild-type viruses with the remaining gene segments derivedfrom a donor virus (often referred to as a master donor virus or MDV)which will have certain desirable characteristics. For example, an MDVstrain may be cold-adapted, and/or temperature sensitive, and/orattenuated, and/or have a high growth rate. According to the presentinvention the virus particles preferably comprise the HA and/or NAproteins of virus strains recommended for seasonal vaccination purposesor of virus strains which have shown to be highly immunogenicspecifically in case of pandemic viruses.

The selection of viruses comprising said surface proteins can be basedon phenotypic, genotypic or antigenic properties which differentiatesaid proteins from HA and NA proteins of helper virus origin.

Phenotypic properties of the HA and NA proteins of helper virus originthat differentiate said proteins from HA and NA proteins of non helpervirus origin, like a selected virus comprises for example differences inthe cleavage site for activation of HA or differs in the stability tolow pH. The helper virus HA may contain a cleavage site that depends onproteolytic activation by a protease different from the proteaseactivating the HA of the vaccine virus. The helper virus may alsoexhibit lower stability to low pH conditions than the vaccine virus.

Selection of viruses containing HA and NA of the vaccine virus may alsobe based on antigenic properties. By using a helper virus of a differentsubtype (e.g. H3N2) than the vaccine virus (e.g. H1N1) growth of thehelper virus can be suppressed by an antiserum specific for helper virussubtype e.g. H3N2. Genotypic characteristics that may be exploited forselection include nucleic acid or amino acid sequence differencesbetween the HA and/or NA segments of helper virus origin and HA and NAproteins of non helper virus origin. Methods are well known in the artto do sequence analysis.

Based on nucleotide sequence differences e.g. siRNAs or anti-senseoligo-nucleotides can be designed specifically for HA and/or NA of thehelper virus. By transfection of these siRNAs or anti-senseoligonucleotides helper virus growth could be suppressed.

One option can be that virus particles comprising HA proteins of helpervirus origin are separated from the candidate virus particles bytreatment with a protease which does not cleave HA protein of helpervirus origin but cleaves and thereby activates the HA protein of thereassortant virus. For example, the protease can be selected from thegroup consisting of trypsin, elastase, chymotrypsin, papain orthermolysin.

For example, the HA protein of the helper virus can be modified to beactivated, e.g. cleavage, by a protease wherein said protease is nottrypsin and whereas the HA protein of the final vaccine virus is cleavedby trypsin. Thereby a simple and applicable selection system isprovided. This can be performed by modifying the cleavage site. The HAsegment a virus strain useful as helper virus can be altered bymutagenesis, such as PCR-mutagenesis, to contain a cleavage site that isproteolytically activated by elastase instead of trypsin. For example,the amino acid sequence surrounding the cleavage site can bePSIQPI/GLFGA (the cleavage site is indicated by /).

To minimise unwanted reversion events codons are chosen in a way that atleast two nucleotide changes per codon are preferably necessary to causea reversion back to the original amino acid.

Alternatively the virus particles comprising HA and NA proteins ofhelper virus origin can be separated from the candidate virus particlescomprising the NA or HA proteins expressed from the linear constructs byproviding low pH conditions. Virus particles cultivated in cell culturefor several passages, specifically in Vero cell culture, show reducedstability towards low pH due to modifications within the HA proteinscompared to strains from clinical isolates comprising wild type HAand/or NA proteins. Thus treatment of the helper virus under low pHconditions, i.e. at a pH between 5.2 and 6.2 leads to reducedpropagation rate of helper virus and therefore to a selection ofcandidate viral particles comprising unmodified HA and/or NA proteins.

As a further alternative embodiment of the invention virus particlescomprising HA and/or NA proteins of helper virus origin are separatedfrom the candidate virus particles by treatment with antiserumcontaining antibodies neutralising or binding to said HA and/or NAproteins of helper virus origin.

A combination of different methods to remove unwanted HA and NA proteinscan also be performed according to the invention.

According to a specific embodiment of the invention, the helper virusparticles can comprise NA protein with reduced activity compared to theNA protein of wild-type virus. The helper virus can in this embodimentlack a functional NA protein, i.e. an NA protein that enables the virusto be released from the host cell, or can lack the NA protein entirely.

According to a further alternative embodiment, the helper viruscomprises the HEF protein of influenza C virus. Influenza C virus hasonly one major surface glyco-protein, HEF (hemagglutinin esterasefusion) which is functionally equivalent to HA protein. The HEF proteincan be activated for example with trypsin or TPCK trypsin as describedin Gao et al. (J. Virol., 2008, 6419-6426) which is incorporated hereinby reference.

Alternatively, modified influenza viruses comprising virus glycoproteinHEF that can be modified by introducing a foreign protease cleavagesite, for example elastase cleavage site, are specifically claimed bythe present invention.

As a further alternative embodiment of the invention virus particlescomprising HEF protein of helper virus origin are removed by treatmentwith antibodies neutralising or binding to said HEF protein.

As a further alternative the helper virus can comprise the HA protein ofa coronavirus. In case of production of influenza A virus, alternativelyHA and/or NA proteins from influenza B origin can be used.

The virus for vaccine production as well as the helper virus canspecifically be of influenza virus origin, more specifically it can bean attenuated influenza virus.

According to a specific embodiment, the influenza virus is an attenuatedinfluenza virus. Specifically the influenza virus comprises deletions ormodifications within the pathogenicity factors inhibiting innate immuneresponse of host cells. The attenuation can exemplarily be derived fromcold-adapted virus strains or due to a deletion or modification withinthe NS1 gene (ANSI virus) as described in WO99/64571 and WO99/64068which are incorporated herein in total by reference. “Modification”refers to a substitution or deletion of one or more nucleic acids ascompared to a wild-type NS1 sequence. Modification within the NS genecan lead to virus particles that are growth deficient in interferoncompetent cells. Growth deficient means that these viruses arereplication deficient as they undergo abortive replication in therespiratory tract of animals. Alternatively, the viruses can comprisedeletion or modification of the PB1-F2 gene.

The method according to the invention can be specifically used forproducing an influenza virus comprising a deletion of functional NS1protein.

According to the invention the helper virus can contain at least 4,preferably at least 5, preferably 6 segments identical to the virus tobe produced. Specifically, these segments are PB1, PB2, PA, NP, M, NS.

Helper virus can be produced by known reverse genetics technologies orby alternative technologies like virus reassortment.

The term “reassortant,” when referring to a virus, indicates that thevirus includes genetic and/or polypeptide components derived from morethan one parental viral strain or source. For example, a 7:1 reassortantincludes 7 viral genomic segments (or gene segments) derived from afirst parental virus, and a single complementary viral genomic segment,e.g., encoding hemagglutinin or neuraminidase, from a second parentalvirus. A 6:2 reassortant includes 6 genomic segments, most commonly the6 internal genes from a first parental virus, and two complementarysegments, e.g., hemagglutinin and neuraminidase, from a differentparental virus.

A method for producing helper virus comprising NS1 deletions wasdescribed by Egorov et al. (1998 J. Virol. 1998 August; 72(8):6437-41;Egorov et al., Vopr. Virusol., 39:201-205). Thereby an H1 influenza Avirus was used as basic virus comprising a temperature sensitivemutation within the NS gene that is further modified to result incompletely deleted NS gene that can only grow in interferon deficientcells.

The present invention also covers a HA polypeptide comprising thesequence of PSIQPIGLFGA (SEQ ID. No. 7).

HA nucleotide sequence comprising following sequence or part thereof isalso covered by the present invention:

(SEQ ID No. 8) AGCAAAAGCAGGGGAAAATAAAAACAACCAAAATGAAAGCAAAACTACTGGTCCTGTTATGTACATTTACAGCTACATATGCAGACACAATATGTATAGGCTACCATGCCAACAACTCAACCGACACTGTTGACACAGTACTTGAGAAGAATGTGACAGTGACACACTCTGTCAACCTACTTGAGGACAGTCACAATGGAAAACTATGTCTACTAAAAGGAATAGCCCCACTACAATTGGGTAATTGCAGCGTTGCCGGATGGATCTTAGGAAACCCAGAATGCGAATTACTGATTTCCAAGGAATCATGGTCCTACATTGTAGAAACACCAAATCCTGAGAATGGAACATGTTACCCAGGGTATTTCGCCGACTATGAGGAACTGAGGGAGCAATTGAGTTCAGTATCTTCATTTGAGAGATTCGAAATATTCCCCAAAGAAAGCTCATGGCCCAACCACACCGTAACCGGAGTATCAGCATCATGCTCCCATAATGGGAAAAGCAGTTTTTACAGAAATTTGCTATGGCTGACGGGGAAGAATGGTTTGTACCCAAACCTGAGCAAGTCCTATGTAAACAACAAAGAGAAAGAAGTCCTTGTACTATGGGGTGTTCATCACCCGCCTAACATAGGGAACCAAAGGGCCCTCTATCATACAGAAAATGCTTATGTCTCTGTAGTGTCTTCACATTATAGCAGAAGATTCACCCCAGAAATAGCCAAAAGACCCAAAGTAAGAGATCAGGAAGGAAGAATCAACTACTACTGGACTCTGCTGGAACCTGGGGATACAATAATATTTGAGGCAAATGGAAATCTAATAGCGCCATGGTATGCTTTTGCACTGAGTAGAGGCTTTGGATCAGGAATCATCACCTCAAATGCACCAATGGATGAATGTGATGCGAAGTGTCAAACACCTCAGGGAGCTATAAACAGCAGTCTTCCTTTCCAGAATGTACACCCAGTCACAATAGGAGAGTGTCCAAAGTATGTCAGGAGTGCAAAATTAAGGATGGTTACAGGACTAAGGAACATCCCATCCATTCAACCCATTGGTTTGTTTGGAGCCATTGCCGGTTTCATTGAAGGGGGGTGGACTGGAATGGTAGATGGGTGGTATGGTTATCATCATCAGAATGAGCAAGGATCTGGCTATGCTGCAGATCAAAAAAGTACACAAAATGCCATTAACGGGATTACAAACAAGGTGAATTCTGTAATTGAGAAAATGAACACTCAATTCACAGCTGTGGGCAAAGAATTCAACAAATTGGAAAGAAGGATGGAAAACTTAAATAAAAAAGTTGATGATGGGTTTCTAGACATTTGGACATATAATGCAGAATTGTTGGTTCTACTGGAAAATGAAAGGACTTTGGATTTCCATGACTTCAATGTGAAGAATCTGTATGAGAAAGTAAAAAGCCAATTAAAGAATAATGCCAAAGAAATAGGAAACGGGTGTTTTGAATTCTATCACAAGTGTAACAATGAATGCATGGAGAGTGTGAAAAATGGAACTTATGACTATCCAAAATATTCCGAAGAATCAAAGTTAAACAGGGAGAAAATTGATGGAGTGAAATTGGAATCAATGGGAGTCTATCAGATTCTGGCGATCTACTCAACTGTCGCCAGTTCCCTGGTTCTTTTGGTCTCCCTGGGGGCAATCAGCTTCTGGATGTGTTCCAATGGGTCTTTGCAGTGTAGAATATGCATCTGAGACCAGAATTTCAGAAATATAAGAAAAAACACCCTTGTTTCTACT

In particular, an HA nucleotide comprising the following sequence isincluded in the present invention:5′-CCATCCATTCAACCCATTGGTTTGTTTGGAGCC-3′ (SEQ ID. 9)

EXAMPLES Example 1 Generation of a Linear H3N2 HA Expression Construct

The HA segment of a Vero cell culture-derived influenza A H3N2 virus wasPCR amplified using the oligonucleotides P1 and P2 (F1 in FIG. 1 a).Subsequently, two DNA fragments (F2 and F3 in FIG. 1) derived frompHW2000 (Hoffmann et al. 2000, Proc Natl Acad Sci USA. 97:6108-13) werefused to the HA PCR product by means of overlapping PCR (see FIG. 1 b).The first DNA fragment (F2) comprises the CMV promoter and the polIterminator, the second one (F3) comprises the human Poll promoter andthe BGH polyA signal. To facilitate generation of the overlapping PCRproducts, oligonucleotides used for HA amplification were extended ontheir 5′ ends in that P1 contains a sequence complementary to the polIterminator and P2 contains a sequence complementary to the polI promoter(see FIG. 1 a). Similarly, the primers P3 and P5 used for generation ofthe fragments F1 and F2 were extended on their 5′ termini to containsequences complementary to the 5′ and 3′ end of the HA (see FIG. 1 a).

Fragments F2 and F3 contain protection sequences derived from sequencedescribed in the pHW2000 backbone. These sequences are not directlyinvolved in transcription of mRNA and vRNA but reduce degradation of thebidirectional expression cassette by exonucleases.

Viral RNA was extracted from a Vero cell culture-derived influenza AH3N2 virus using a Qiagen ViralAmp kit and reverse transcribed using theUni12 oligonucleotide as described previously (Hoffmann et al. 2001,Arch Virol. 146:2275-89).

The HA segment was amplified with the oligonucleotides shown in thetable 1 using a mixture of Pfu Turbo DNA polymerase and Taq DNApolymerase:

TABLE 1 P1 5′-CGAAGTTGGGGGGG

-3′ (SEQ ID No. 1) P2 5′-GCCGCCGGGTTATT

-3′ (SEQ ID No. 2)

Nucleotides corresponding to the H3 sequence are shown in italic boldletters, nucleotides homologous to the polI terminator (P1) and the polIpromoter (P2) are shown in standard capital letters.

The HA F4 PCR product was purified using a Qiaquick PCR Purification kit(Qiagen). PCR fragments F2 and F3 were amplified from pHW2000 plasmidDNA with the primer pairs P3+P4 and P5+P6 (see table 2 and FIG. 1 a),respectively using a mixture of Pfu Turbo DNA polymerase and Taq DNApolymerase. PCR products F2 and F3 were purified using a QIAquick PCRPurification kit (Qiagen)

TABLE 2 P3 5′-

CCCCCCCAACTTCGGAGGTC-3′ (SEQ ID No. 3) P45′-GGGGTATCAGGGTTATTGTCTCATGAGCGGATAC-3′ (SEQ ID No. 4) P5 5′-

AATAACCCGGCGGCCCAAAATGC-3′ (SEQ ID No. 5) P65′-CCCCTTGGCCGATTCATTAATGCAGCTGGTTC3′ (SEQ ID No. 6)

For P3 and P5 nucleotides corresponding to the H3 sequence are shown initalic bold letters, nucleotides complementary to pHW2000 are shown instandard capital letters.

For P4 and P6 all nucleotides except the four nucleotides at the 5′ endscorrespond to pHW2000.

For generation of the full length PCR product (F4) containing the HA,the CMV promoter, the polI terminator, the polI promoter and the BGHpolyA signal, fragments F1, F2 and F3 were combined and amplified byoverlapping PCR with the primers P4 and P6 using a mixture of Pfu TurboDNA polymerase and Taq DNA polymerase.

FIG. 1 shows a schematic diagram of the generation of linearbidirectional expression constructs.

FIG. 1 a) schematically discloses Fragments F1, F2 and F3 generatedseparately by PCR amplification.

Fragment F1 contains the respective viral segment and containsextensions complementary to the polI promoter and polI terminator.Fragment F2 contains the CMV promoter and the polI terminator as well asan extension complementary to the respective viral segment. Fragment F3contains the polI promoter and the BGH poly adenylation signal as wellas an extension complementary to the respective viral segment.Oligonucleotides P1 and P2 used for PCR amplification of F1 fragmentsare complementary to the respective viral segment. P1 contains a 5′extension complementary to the polI terminator, P2 contains a5′extension complementary to the polI promoter.

Oligonucleotides P3 and P4 are used for PCR amplification of F2fragments with P3 containing a 5′extension complementary to therespective viral segment. Oligonucleotides P5 and P6 are used for PCRamplification of F3 fragment with P5 containing a 5′extensioncomplementary to the respective viral segment. Protection sequences arederived from the pHW2000 backbone and do not contain sequences directlyinvolved in mRNA or vRNA transcription.

Example 2 Generation of an Elastase-Dependent Helper Virus

The HA segment of a influenza A/New Caledonia/20/99-like (H1N1) strainis altered by PCR-mutagenesis to contain a cleavage site that isproteolytically activated by elastase instead of trypsin. The amino acidsequence surrounding the cleavage site is changed from PSIQSR/GLFGA toPSIQPI/GLFGA (the cleavage site is indicated by /). Analogous to example1, 10-20 μg linear bidirectional expression construct F4 are generatedby PCR and purified using a Qiaquick kit (Qiagen) and subsequently via aQiagen Endofree Plasmid kit.

Vero cells are maintained in DMEM/F12 medium containing 10% foetal calfserum and 1% Glutamax-I supplement at 37° C. and 5% CO2.

For virus generation the modified F4 HA DNA fragment is used alone ortogether with four protein expression plasmids coding for PB1, PB2, PAand NP for transfection of Vero cells. 24 h after transfection cells areinfected at an MOI of 0.001 to 1 with an influenza A IVR-116 strain thatdoes not express a functional NS1 (IVR-116-deINS1). Following infection,to support virus replication, Vero cells are cultured in serum-freemedium (Opti-Pro; Invitrogen) in the presence of 5 μg/ml elastase. Assoon as 50-100% CPE is observed the rescued elastase-dependentIVR-116-deINS1 virus (IVR-116-deINS1-EL) is frozen or plaque-purified onVero cells.

Example 3 Generation of an Influenza A H3N2 Reassortant Virus by Usingan Elastase-Dependent H1N1 Helper Virus

Linear bidirectional expression constructs (F4) for the HA and NAsegments of a A/Brisbane/10/2007 (H3N2)-like virus are generated by PCRas described in example 1. Following purification as described inexample 2 the HA and NA F4 PCR products are used alone or together withfour protein expression plasmids coding for PB1, PB2, PA and NP fortransfection of Vero cells. 24 h after transfection cells are infectedat an MOI of 0.001 to 1 with influenza A IVR-116-deINS1-EL virus (helpervirus). Following infection cells are incubated in serum-free medium(Opti-Pro; Invitrogen) in the presence of 5 μg/ml trypsin. As soon as10-100% CPE is observed virus is harvested. A selective passage isperformed by treating the viral harvest for 24 h at 4° C. withappropriate concentrations (e.g. 10% v/v) of antisera (pretreated withneuraminidase from Vibrio cholerae) or of a purified IgG preparationspecific for A/New Caledonia/20/99 HA and NA to neutralise helper virus.Vero cells are then incubated for 30 min at RT with pretreated virus,washed with PBS and subsequently incubated at 37° C. in serum-freemedium containing 5 μg/ml trypsin. Optionally, purified IgG specific forA/New Caledonia/20/99 HA and NA may be added to the culture medium. Assoon as 10-100% CPE is observed virus is harvested and a secondselective passage is performed.

Upon development of CPE virus is frozen or plaque-purified.

Example 4 Generation of an Influenza A H3N2 Reassortant Virus by Usingan Elastase-Dependent H1N1 Helper Virus in Combination with Low pHTreatment

Linear bidirectional expression constructs (F4) for the HA and NAsegments of a A/Brisbane/10/2007 (H3N2)-like virus are generated by PCRas described in example 1. Following purification as described inexample 2 the HA and NA F4 PCR products are used alone or together withfour protein expression plasmids coding for PB1, PB2, PA and NP fortransfection of Vero cells. 24 h after transfection cells are infectedat an MOI of 0.001 to 1 with influenza A IVR-116-deINS1-EL virus (helpervirus). Following infection cells are incubated in serum-free medium(Opti-Pro; Invitrogen) in the presence of 5 μg/ml trypsin. As soon as10-100% CPE is observed virus is harvested. Viral harvest is thendiluted 1:1 with buffer containing 150 mM NaCl, and 50 mM MES pH 5.4-6.2and incubated for 30 min at 37° C. to preferentially inactivate helpervirus HA. Following pH neutralisation a selective passage can thenperformed by incubating the viral harvest for 24 h at 4° C. withappropriate concentrations (e.g. 10% v/v) of antisera (pretreated withneuraminidase from Vibrio cholerae) or of a purified IgG preparationspecific for A/New Caledonia/20/99 HA and NA to neutralise helper virus.Vero cells are then incubated for 30 min at RT with pretreated virus,washed with PBS and subsequently incubated at 37° C. in serum-freemedium containing 5 μg/ml trypsin. Optionally, purified IgG specific forA/New Caledonia/20/99 HA and NA may be added to the culture medium. Assoon as 10-100% CPE is observed virus is harvested and a secondselective passage is performed.

Upon development of CPE virus is frozen or plaque-purified.

Example 5 Generation of an Influenza A H1N1 Reassortant Virus by Usingan Elastase-Dependent H3N2 Helper Virus

Linear bidirectional expression constructs (F4) for the HA and NAsegments of a A/New Caledonia/20/99 (H1N1)-like virus are generated byPCR as described in example 1. Following purification as described inexample 2 the HA and NA F4 PCR products are used alone or together withfour protein expression plasmids coding for PB1, PB2, PA and NP fortransfection of Vero cells. 24 h after transfection cells are infectedat an MOI of 0.001 to 1 with an elastase-dependent influenzaA/Wisconsin/67/05 (H3N2)-like virus (helper virus). Following infectioncells are incubated in serum-free medium (Opti-Pro; Invitrogen) in thepresence of 5 μg/ml trypsin. As soon as 10-100% CPE is observed virus isharvested. A selective passage is performed by treating the viralharvest for 24 h at 4° C. with appropriate concentrations (e.g. 10% v/v)of antisera (pretreated with neuraminidase from Vibrio cholerae) or of apurified IgG preparation specific for A/Wisconsin/67/05 HA and NA toneutralise helper virus. Vero cells are then incubated for 30 min at RTwith pretreated virus, washed with PBS and subsequently incubated at 37°C. in serum-free medium containing 5 μg/ml trypsin. Optionally, purifiedIgG specific for A/Wisconsin/67/05 HA and NA may be added to the culturemedium. As soon as 10-100% CPE is observed virus is harvested and asecond selective passage is performed.

Upon development of CPE virus is frozen or plaque-purified.

1. A method for production of negative stranded segmented RNA viruscomprising the steps of: a) providing a linear expression construct freeof any amplification and/or selection sequences, which constructcomprises an RNA polymerase I (polI) promoter and a polI terminationsignal, both inserted between an RNA polymerase II (polII) promoter anda polyadenylation signal, which construct further comprises a HA and/ora NA gene segment inserted between the polI promoter and the polItermination signal, b) transfecting a host cell with said linearexpression construct, c) infecting said host cells with a helper virushaving helper virus HA and/or NA proteins, d) cultivating said host cellto propagate progeny virus particles, e) selecting virus particles,which contain: (i) the HA and/or NA proteins derived from the linearexpression construct, but not (ii) the helper virus HA and NA proteins,or segments thereof, wherein said selection is based on phenotypic,genotypic or antigenic properties of the HA and/or NA proteins.
 2. Amethod according to claim 1, wherein said linear expression constructencodes proteins selected from the group consisting of PB1, PB2, PA, NS,M, and NP.
 3. A method according to claim 1, wherein progeny virusparticles comprising HA protein derived from the helper virus areseparated from candidate virus particles by treating progeny virusparticles with a protease, and wherein the protease does not cleave theHA protein derived from the helper virus but cleaves the HA protein ofthe candidate virus particles
 4. A method according to claim 3, whereinthe protease is selected from the group consisting of trypsin, elastase,chymotrypsin, and papain.
 5. A method according to claim 1, wherein theHA protein of the helper virus is modified to be cleaved by a protease,wherein said protease is not trypsin.
 6. A method according to claim 1,wherein progeny virus particles comprising HA and NA proteins of helpervirus origin are separated from candidate virus particles by providinglow pH conditions.
 7. A method according to claim 1, wherein progenyvirus particles comprising HA and/or NA proteins of helper virus originare separated from candidate virus particles by contacting the progenyvirus with antibodies binding said HA and/or NA proteins.
 8. A methodaccording to claim 1, wherein the helper virus particles comprise NAprotein with reduced activity, or lack a functional NA protein.
 9. Amethod according to claim 1, wherein the helper virus comprises the HEFprotein of influenza C virus.
 10. A method according to claim 9 whereinHEF protein of the helper virus is modified to be cleaved by a proteasewherein said protease is not trypsin.
 11. A method according to claim 1,wherein the helper virus comprises the HA protein of a coronavirus. 12.A method according to claim 1, wherein said progeny virus particle is aninfluenza virus particle.
 13. A method according to claim 12, whereinsaid progeny virus particle and/or said helper virus particle is anattenuated influenza virus particle.
 14. A method according to claim 13,wherein said progeny virus particle and/or said helper virus particlecomprises a deletion or modification within the NS1 gene.
 15. A methodaccording to claim 13, wherein the helper virus comprises a modified ordeleted NS gene and is growth deficient in interferon competent cells.16. A method according to claim 1, wherein the helper virus contains atleast 4 segments identical to the candidate virus.
 17. An HA polypeptidefragment comprising the sequence PSIQPIGLFGA (SEQ ID. NO. 7).
 18. An HAnucleotide sequence fragment comprising the sequenceCCATCCATTCAACCCATTGGTTTGTTTGGAGCC (SEQ ID. NO. 9).
 19. A methodaccording to claim 16, wherein the helper virus contains at least 5segments identical to the candidate virus.
 20. A method according toclaim 19, wherein the helper virus contains at least 6 segmentsidentical to the candidate virus.
 21. A method according to claim 1,wherein the absence of helper virus HA and NA proteins is determined byanalysis of the nucleic acid or amino acid sequence.